Insulin-like growth factor 1 (IGF-1) is an anabolic hormone produced in the liver that is stimulated by the growth hormone (GH). GH binds to the GH receptors on the hepatocyte cell membrane and triggers an unknown mechanism that synthesizes and releases IGF-1 into the blood. The normal levels of IGF-1 are between 120-400 ng/ml. IGF-1 is involved in the regulation of cell proliferation and differentiation of a wide variety of cell and tissue types, and plays an important role in tissue renewal and repair. Because of these important applications of IGF-1 in the body, people who suffer IGF-1 deficiency have many harmful side effects. For example of this is patients with liver cirrhosis who have a reduction of the GH receptor in the hepatocytes and the diminished synthesis of the liver parenchyma causes a significant decrease of IGF-I levels in the blood (20 ng/ml and frequently to undetectable levels) with different systemic problems like muscle atrophy, osteopenia, hypogonadism, protein-calorie malnutrition, weight loss, and many others. Recent studies showed that treatments with low doses of IGF-I help to induce significant improvements in nutritional status, intestinal absorption, hypogonadism, and liver functions in rats with liver cirrhosis. Replacement therapy with IGF-1 in liver cirrhosis patients requires daily doses of 1.5 to 2 mg. Thus, a single patient would need to consume about 600 mg/year. However, the IGF-1 treatment is very expensive, $30,000/mg. Besides, as described above, IGF-1 is used in treatment of dwarfism, diabetes, osteoporosis, starvation, and hypercatabolism.
The insulin-like growth factors I and II (IGF-I and IGF-II, respectively) mediate multiple effects in vivo, including cell proliferation, cell differentiation, inhibition of cell death, and insulin-like activity (reviewed in Clark and Robinson, Cytokine Growth Factor Rev., 7: 65-80 (1996); Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)). Most of these mitogenic and metabolic responses are initiated by activation of the IGF-I receptor, an .alpha..sub.2.beta..sub.2-heterotetramer closely related to the insulin receptor (McInnes and Sykes, Biopoly., 43; 339-366 (1997); Ullrich et al., EMBO J., 5: 2503-2512 (1986)). Both proteins are members of the tyrosine kinase receptor superfamily and share common intracellular signaling cascades (Jones and Clemmons, supra). IGF-insulin hybrid receptors have been isolated, but their function is unknown. The IGF-I and insulin receptors bind their specific ligands with nanomolar affinity. IGF-I and insulin can cross-react with their respective non-cognate receptors, albeit at a 100-1000-fold lower affinity (Jones and Clemmons, supra). The crystal structure describing part of the extracellular portion of the TGF-I receptor has recently been reported (Garrett et al., Nature, 394: 395-399 (1998)). When referring to IGF-1 in this application it should be understood that the aspects of this invention may utilize all IGF-1 and all variants of IGF-1 which have been described in the art.
IGF-I is a single-chain 70-amino-acid protein with high homology to proinsulin. Unlike the other members of the insulin superfamily, the C region of the IGF's is not proteolytically removed after translation. The solution NMR structures of IGF-I (Cooke et al., Biochemistry, 30: 5484-5491 (1991); Hua et al., J. Mol. Biol., 10 259: 297-313 (1996)), mini-IGF-I (an engineered variant lacking the C-chain; DeWolf et al., Protein Science, 5: 2193-2202 (1996)), and IGF-II (Terasawa et al., EMBOJ., 13: 5590-5597 (1994); Torres et al., J. Mol. Biol. 248: 385-401 (1995)) have been reported. It is generally accepted that distinct epitopes on IGF-I are used to bind receptor and binding proteins. It has been demonstrated in animal models that receptor-inactive IGF mutants are able to displace endogenous IGF-I from binding proteins and hereby generate a net IGF-I effect in vivo (Loddick et al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al., Biochemistry, 37: 8870-8878 (1998)). While residues Y24, Y29, Y31, and Y60 are implicated in receptor binding, IGF mutants thereof still bind to IGFBPs (Bayne et al., J. Biol. Chem., 265: 15648-15652 (1990); Bayne et al., J. Biol. Chem., 264: 11004-11008 (1989); Cascieri et al., Biochemistry, 27: 3229-3233 (1988); Lowman et al., supra.
Other IGF-I variants have been disclosed. For example, in the patent literature, WO 96/33216 describes a truncated variant having residues 1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-I superagonists which are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BC.sup.n,A wherein B is the B domain of IGF-I or a functional analog thereof, C is the C domain of IGF-I or a functional analog thereof, n is the number of amino acids in the C domain and is from about 6 to about 12, and A is the A domain of IGF-I or a functional analog thereof.
Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-I, three of which have reduced affinity to the Type 1 IGF receptor. These mutants are: (Phe.sup.23, Phe.sup.24, Tyr.sup.25) IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu.sup.24) IGF-I and (Ser.sup.24) IGF-I (which have a lower affinity than IGF-I to the human placental Type 1 IGF receptor, the placental insulin receptor, and the Type 1 IGF receptor of rat and mouse cells), and desoctapeptide (Leu.sup.24) IGF-I (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF-I, which has lower affinity than (Leu.sup.24) IGF-I for the Type 1 receptor and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum binding proteins.
Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses three structural analogs of IGF-I: (1-62) IGF-I, which lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27, Gly.sup.4, 38-70) IGF-I, in which residues 28-37 of the C region of IGF-I are replaced by a four-residue glycine bridge; and (1-27, Gly.sup.4, 38-62) IGF-I, with a C region glycine replacement and a D region deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly.sup.4 mutant of Bayne et al., supra, Vol. 264. U.S. Pat. No. 5,714,460 refers to using IGF-I or a compound that increases the active concentration of IGF-I to treat neural damage.
Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses three IGF-I analogs in which specific residues in the A region of IGF-I are replaced with the corresponding residues in the A chain of insulin. The analogs are: (Ile.sup.41, Glu.sup.45, Gln. sup.46, Thr.sup.49, Ser.sup.50, Ile.sup.51, Ser. sup.53, Tyr.sup.55, Gln.sup.56) IGF-I, an A chain mutant in which residue 41 is changed from threonine to isoleucine and residues 42-56 of the A region are replaced; (Thr.sup.49, Ser.sup.50, Ile.sup.51) IGF-I; and (Tyr.sup.55, Gln.sup.56) IGF-I.
WO 94/04569 discloses a specific binding molecule, other than a natural IGFBP, that is capable of binding to IGF-I and can enhance the biological activity of IGF-I. WO 98/45427 published Oct. 15, 1998 and Laowman et al., supra, disclose IGF-I agonists identified by phage display. Also, WO 97/39032 discloses ligand inhibitors of IGFBP's and methods for their use.
There are various forms of human insulin on the market that differ in the duration of action and onset of action, but have the native human sequence. Jens Brange, Galenics of Insulin, The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (Springer-Verlag, N.Y., 1987), page 17-40. Regular insulin is a clear neutral solution that contains hexameric insulin. It is short acting, its onset of action occurs in 0.5 hour after injection and duration of action is about 6-8 hours. NPH (Neutral Protamine Hagedorn) insulin, also called Isophane Insulin, is a crystal suspension of insulin-protamine complex. These crystals contain approximately 0.9 molecules of protamine and two zinc atoms per insulin hexamer. Dodd et al., Pharmaceutical Research, 12: 60-68 (1995). NPH-insulin is an intermediate-acting insulin; its onset of action occurs in 1.5 hours and its duration of action is 18-26 hours. 70/30 insulin is composed of 70% NPH-insulin and 30% Regular insulin There are also Semilente insulin (amorphous precipitate of zinc insulin complex), UltraLente insulin (zinc insulin crystal suspension), and Lente insulin (a 3:7 mixture of amorphous and crystalline insulin particles). Of the various types of insulins available, NPH-, 70/30, and Regular insulin are the most widely used insulins, accounting for 36%, 28%, and 15%. respectively, of the insulin prescriptions in 1996.
The use of recombinant DNA technology and peptide chemistry have allowed the generation of insulin analogs with a wide variety of amino acid substitutions, and IGF-like modifications to insulin have been made for the purpose of modifying insulin pharmacokinetics (Brange et al., Nature, 333: 679 (1988); Kang et al., Diabetes Care, 14: 571 (1991); DiMarchi et al., “Synthesis of a fast-acting insulin analog based upon structural homology with insulin-like growth factor-I,” in: Peptides: Chemistry and Biology, Proceedings of the Twelfth American Peptide Symposium, J. A. Smith and J. E. Rivier, eds. (ESCOM, Leiden, 1992), pp. 26-28; Weiss et al., Biochemistry, 30: 7373 (1991); Howey et al., Diabetes, 40: (Supp 1) 423A (1991); Slieker and Sundell, Diabetes, 40: (Supp 1) 168A (1991); Cara et al., J. Biol. Chem., 265: 17820 (1990); Wolpert et al., Diabetes, 39: (Supp 1) 140A (1990); Bornfeldt et al., Diabetologia, 34: 307 (1991); Drejer, Diabetes/Metabolism Reviews, 8: 259 (1992); Slieker et al., Adv. Experimental Med. Biol., 343; 25-32 (1994)). One example of such an insulin analog is Humalog.™ insulin (rapid-acting monomeric insulin solution, as a result of reversing the Lys (B28) and Pro (B29) amino acids on the insulin B-chain) that was recently introduced into the market by Eli Lilly and Company. A review of the recent insulin mutants in clinical trials and on the market is found in Barnett and Owens, Lancet, 349: 47-51 (1997).
Additionally, a variant designated (1-27, gly.sup.4, 38-70) hIGF-I, wherein residues 28-37 of the C region human IGF-I are replaced by a four-residue glycine bridge, has been discovered that binds to IGFBP's but not to IGF receptors (Bar et al., Endocrinology, 127: 3243-3245 (1990)).
Currently, most of the IGF-1 that is available is expressed in E. coli. The main problem with this expression system is that E. coli cannot produce the mature IGF-1 because E. coli does not form disulfide bonds in the cytoplasm. The polypeptide has to be targeted in the periplasm to form disulfide bonds. The cost of IGF-1 production increases when the protein is in the periplasm because it is harder to purify and the IGF-1 is not properly folded because the disulfide bonds were not formed. Transgenic plants are good expression systems for large-scale production of recombinant proteins at industrial levels. Plant systems have many advantages, such as: the low cost of growing plants on a large scale, the availability of natural protein storage organs, and the established practices for their efficient harvesting, transporting, storing, and processing. It has been estimated that the cost of producing recombinant proteins in plants could be 10 to 50 fold lower than producing the same protein by E. coli via fermentation. A drawback of the plant systems is the low expression levels of recombinant proteins. In general, proteins produced in nuclear transgenic plants are relatively low, mostly less than 1% of the total soluble protein. Some examples of these proteins are human serum albumin 0.02%, hemoglobin 0.05%, and erythropoietin 0.0026% of total soluble protein. Also, a synthetic gene coding for the human epidermal growth factor was expressed only up to 0.001% of total soluble protein in transgenic tobacco. One of the reasons for low expression levels is that in nuclear transformation the gene is inserted randomly resulting in position effect or the expression of transgene is silenced. To avoid these problems the proteins can be expressed in the plastid. Chloroplast transformation is a recent technique that has overcome limitations of nuclear transformation, such as the low expression levels of recombinant proteins and transgene containment. A good example of the success of this technique is the high accumulation of the cryIIA protein, up to 47% of total cellular protein. Another advantage is that the presence of proteins in chloroplasts that facilitate posttranslational modifications, including the folding and assembly of prokaryotic and eukaryotic proteins. An example of this is the integration of the native Cholera Toxin B subunit into chloroplast genomes, and its assembly as functional oligomers was successfully achieved in transgenic tobacco chloroplast reaching an accumulation of 4.1% of total soluble protein.
One aspect of this invention is to create recombinant DNA vectors in order to enhance and show variable expression levels of the human IGF-1 protein via the plastid, and to study the difference in expression levels between the synthetic and the native human IGF-1 genes.
Unique to plants is the ability to regenerate whole plants from cells or tissues. This totipotency has many practical benefits: for example, plants propagated by seed can be cultured in vitro to yield thousands of identical plants (Bhojwani, 1990). In particular, tobacco is the easiest plant to genetically engineer and is widely used to test suitability of plant-based systems for bioproduction of recombinant proteins. Tobacco is an excellent biomass producer (in excess of 40 tons leaf fresh weight/acre based on multiple mowings per season) and a prolific seed producer (up to one million seeds produced per plant), thus hastening the time in which a product can be scaled up and brought to market (Cramer et al., 1998). In general, plant systems are more economical than industrial facilities using fermentation or bioreactor systems and the technology is already available for harvesting and processing plants and plant products on a large scale (Daniell et al., 2001a). Plant-derived products are less likely to be contaminated with human pathogenic microorganisms than those derived from animal cells because plants don't act as hosts for human infectious agents (Giddings et al., 2000).
Recombinant proteins expressed in plant cells are naturally protected from degradation when taken orally (Kong et al., 2001). Oral delivery is highly desirable for drug treatment (Gomez-Orellan and Paton, 1998).
The genetic information of plants is distributed among three cellular compartments: the nucleus, the mitochondria, and the plastids and each of these carries its own genome and expresses heritable traits (Bogorad, 2000). Transformation of the plant nucleus is routine in many species and there are a variety of techniques for delivering foreign DNA to the plant nuclear genome (Hager and Bock, 2000). However, recombinant protein expression in plants by nuclear transformation have been dismally low, with most levels much less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified (Daniell et al., 2002). Also incorporated by reference into this application is the utility application, based off of U.S. Provisional Application No. 60/393,651, and filed simultaneously with this application. Still another application, PCT/US02/41503, filed on Dec. 26, 2002, is also incorporated by reference into this application. In a general sense these applications describe in detail somatic embryogenosis for the construction of edible vaccines.
The plastids of plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. In general, plant cells contain 500-10,0000 copies of a small 120-160 kilobase circular plastid genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which can result in very high levels of foreign gene expression.
The modern chloroplast of plants has retained a largely prokaryotic system of gene organization and expression, with the eukaryotic nuclear genome exerting significant regulatory control (Hager and Bock, 2000). Signaling pathways have evolved to coordinate gene expression between the chloroplast and the nuclear-cytosolic compartments during chloroplast development and in response to environmental factors such as light (Zerges, 2000). Illuminated chloroplasts possess extraordinarily high rates of transcription and translation that is tissue-specific due to regulation via untranslated regions of chloroplast-encoded mRNAs. Although communication between the chloroplast and the nucleus exist, these membrane-separated genetic systems have their own distinct environmental milieu containing different proteins, proteases and mechanisms of action. Unique features of the photosynthetic plastid enable genetic engineering of the chloroplast to overcome major limitations of plant nuclear transformation technology. One major concern with the genetic modification (GM) of plants is the possibility of the escape of foreign genes through pollen dispersal from transgenic plants to sexually compatible weedy relatives or to pathogenic microbes in the soil (Daniell, 2002). Such gene transfers could potentially result in the emergence of “superweeds” able to resist certain herbicides thereby undermining the benefits of GM crops (Daniell, 2002). However, genes in the chloroplasts of higher plants are generally transmitted only by the maternal parent, which means that chloroplast genes are not present in the pollen (Bogorad, 2000). Therefore, a foreign gene introduced by genetic engineering of the chloroplast genome could not transfer to genetically compatible weeds. This uniparental or maternal inheritance provides the gene containment necessary for keeping foreign genes sequestered in target plants and preventing gene flow among crops and weeds (Daniell, 2002).
Another remarkable feature of the plastid genome is its extremely high ploidy level: a single tobacco leaf cell may contain as many as 100 chloroplasts, each harboring approximately 100 identical copies of the plastid genome, resulting in an extraordinarily high ploidy degree of up to 10,000 plastid genomes per cell (Bogorad, 2000). Because of the very high ploidy level of the plastid genome, very high expression levels can be achieved. For example, the Bacillus thuringiensis (Bt) Cry2Aa2 protein accumulated as cuboidal crystals in transgenic chloroplasts and reached a level of 45.3% of the tsp in mature leaves (De Cosa et al., 2001).
For transformation of chloroplasts in plants, particle bombardment is used to introduce transgenes into leaf chloroplasts and stable transformation requires that all 10,000 chloroplast copies be uniformly converted (Bock and Hagemann, 2000). Securing genetically stable lines of plants with transgenic chloroplast requires every chloroplast to carry the inserted gene (Bogorad, 2000). This homoplasmic state is achieved through amplification and sorting of transgenic chloroplasts with the elimination of the wild-type copies on selective medium. The integration of cloned plastid DNA into the plastid genome occurs through site-specific homologous recombination in plants such as in tobacco N. tabacum and excludes the foreign vector DNA (Kavanagh et al., 1999). In contrast, nuclear transformation experiments in higher plants frequently suffer from epigenetic gene-silencing mechanisms resulting in inconsistent and unstable gene expression or complete loss of transgenic activity (Hager and Bock, 2000). The nuclear genome has mechanisms to effectively inactivate genes when regulatory sequences are inserted in a repetitive pattern and this occurs because integration of transgenes into the nuclear genome is random and through non-homologous recombination (Daniell and Dhingra, 2002). Random integrations of transgenes also means that the final location of the inserted gene may be in region of the nuclear genome that is not highly transcribed. As a consequence, nuclear expression levels vary in different transgenic lines and these differences are due the inserted gene's random position in the nuclear genome. Neither gene silencing nor position effects have been observed in genetically engineered chloroplasts (Daniell, and Dhingra, 2002).
Another major advantage of chloroplast engineering is the expression of multiple transgenes as operons due to efficient translation of polycistronic messenger RNAs (De Cosa et al., 2001). Genetic engineering has now moved from introducing single gene traits to coding for complete metabolic pathways, bacterial operons, and biopharmaceuticals that require assembly of complex multisubunit proteins (Daniell, 2002).
Disulfide bonds are common to many extracellular proteins because they stabilize the native conformation by lowering the entropy of the unfolded form (Abkevich and Shakhnovich, 2000). Most proteins need to be folded correctly for the protein to function properly and remain in solution. Eukaryotic secretory proteins are normally routed through the endoplasmic reticulum where disulfide bond formation occurs. Experiments show that chloroplasts have the machinery needed to fold complex eukaryotic secretory proteins in the soluble chloroplast stroma compartment. The activities of several chloroplast enzymes involved in the anabolic processes of carbon assimilation are enhanced or triggered by light through a signaling system called the ferredoxin-thioredoxin system (Ruelland and Miginiac, Maslow, 1999). Two correct disulfide bonds were formed in the tobacco chloroplast expression of human somatotropin (Staub et al., 2000). In another study, binding assays confirmed that chloroplast-synthesized cholera toxin of Vibrio cholera (CTB) bound intestinal receptors indicating that correct folding and disulfide bond formation had occurred (Daniell et al., 2001). The light signal sensed by chlorophyll is transferred via the photosynthetic electron flow to proteins called thioredoxins, which are very efficient in thio-disulfide interchanges with various protein disulfides (Ruelland and Miginiac-Maslow, 1999). Another mechanism for the simple, reversible activation of genes that regulate expression in the chloroplast is the Protein Disulfide Isomerase (PDI) system composed of chloroplast polyadenylate-binding proteins that specifically bind to the 5′UTR of the psbA mRNA and are modulated by redox status through PDI (Kim and Mayfield, 1997). The ability of chloroplasts to form disulfide bonds and properly fold foreign proteins eliminates a major part of the costly downstream processing.
Expression of functional human somatotropin in transgenic tobacco chloroplasts established that chloroplasts are capable of proper folding of human proteins with disulphide bonds (Staub et al., 2000). The ability to express multiple genes in a single transformation event (Daniell and Dhingra, 2002; De Cosa et al., 2001), accumulation of exceptionally large quantities of foreign proteins (De Cosa et al., 2001), successful engineering of tomato chromoplasts for high level transgene expression in fruits (Ruf et al., 2001, or carrots (Kumar et al., 2003), coupled to hyper-expression of vaccine antigens (Daniell et al., 2001b), and the use of plant derived antibiotic free selectable markers (Daniell et al., 2001c), augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most. The term “edible vaccine” or “oral delivery” as used herein refers to a substance which may be given orally which will elicit a protective immunogenic response in a mammal.
Good recombinant systems are still not available for many human proteins that are expensive to purify or highly susceptible to proteolytic degradation. It is known that traditional purification of biopharmaceuticals proteins using columns accounts for 30% of the production cost and 70% of the set up cost (Petrides et al., 1995). Proteolytic degradation is another serious concern for industrial bioprocessing. The increasing production of proteins in heterologous hosts through the use of recombinant DNA technology has brought this problem into focus; heterologous proteins appear to be more prone to proteolysis (Enfors, 1992). Recombinant proteins are often regarded by a cell as foreign and therefore degraded much faster than most endogenous proteins (Rozkov et al., 2000). Proteolytic stability of recombinant proteins is a significant factor influencing the final yield. In view of these limitations, the Applicant has developed a more efficient method for producing a recombinant biopharmaceutical protein, such as IGF-1 production, which may be used as a model system to enrich or purify biopharmaceutical proteins from transgenic plants, which are highly susceptible to proteolytic degradation. It should be understood that when referring to IGF-1, the term included all variants of IGF-1 which are known in the art.
To date the no one has successfully transformed the plastid genome with IGF to create a delivery system that is easily administered and that stimulates both arms of the immune system without the severe side effects experienced by patients in current IGF treatments. In addition, until the Applicant's discovery, all production vehicles (E. coli, nuclear plant genomes, etc. . . . ) have failed to provide a cost effective and functional IGF, which can be orally administered without the side effects, i.e. human pathogens that are associated with the current production vehicles. In view of these limitations the Applicant developed a system for the expression of biopharmaceutical proteins, such as IGF, via the chloroplast genome in order to provide a feasible means of overproducing this increasingly useful therapeutic drug as well as addressing current concerns with the present methods of delivery and production.